direct PCR from E. coli

Dr. Duncan Clark duncan at genesys.demon.co.uk
Fri May 3 07:52:52 EST 1996


In article <31861C8C.3719 at macollamh.ucd.ie>, Geraldine Butler
<gbutler at MACOLLAMH.UCD.IE> writes
>I know this has been posted several times, but I don't seem to be able 
>to interact with the bionet search engine.... could someone please 
>post or email me a simple method for direct PCR with E. coli colonies?
>
>Thanks,
>
>gbutler at ucd.ie

Tried and well, well tested here, is to do the following.

1.      Toothpick colony, grid then put toothpick in 20ul water in 0.5ml
eppendorf.

2.      When all colonies picked, swill toothpicks around, remove and
place tubes on PCR machine for 5mins at 100C.

3.      Take 1ul (no microfuging) and use in 50ul PCR for 25 cycles. 

4.      5ul is enough to see a very substantial band.

5.      Used from 200bp up to 2.5kb, Universal/Reverse primers or
others.  

6.      For checking mutants we take 5ul from PCR and in a total of 25ul
including appropriate RE buffer do an RE digest. Run out all the digest.

7.      Protocol has been used for well over 1000 colony screenings.

Good luck

Duncan 
  
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